Dr. Sheldon Chen is currently conducting his research in the laboratory of Dr. Faud N. Ziyadeh at the University of Pennsylvania, which boasts a world-renowned biomedical community and provides state- of- the-art facilities, superb human resources, and outstanding technical support. By completing the proposed project, Dr. Chen hopes to acquire new technical expertise, improve his ability to synthesize data into a credible paradigm, hone his critical thinking skills, and afterwards pursue novel or related research that expands upon his findings. After an additional year of mentored fellowship research training, he will join the faculty of the Renal Division at the University of Pennsylvania, where he hopes to mature into a fully-independent academic physician-scientist. Diabetic kidney disease remains the number one cause of renal failure in the United States. As the defining metabolic abnormality in diabetes mellitus, high blood glucose undoubtedly plays an important role in the pathogenesis of diabetic nephropathy. Our laboratory has discovered that Transforming Growth Factor-beta (TGF-beta) mediates much of the injuries effect of hyperglycemia, and the two metabolic factors may conspire to cause even greater renal damage. The podocyte is a highly specialized epithelial cell in the glomerulus that synthesizes part of the glomerular basement membrane (GBM) and regulates the macromolecular permeability across the glomerular filtration barrier. Podocyte dysfunction secondary to the deleterious environment of the diabetic milieu may contribute to the GBM thickening and increased macromolecular permeability leading to proteinuria in diabetic glomerulopathy. Recent cell culture advances have allowed us to study the in vitro podocyte in its fully differentiated state. We propose to examine the effects of high glucose and exogenous TGF-beta on the cultured, differentiated mouse podocyte with respect to the altered expression of type IV collagen and vascular endothelial growth factor (VEGF), two proteins that are likely involved in the pathophysiology of GBM thickening and diabetic proteinuria. Northern analysis will be used to quantitate the gene expression, and immunoblotting and ELISA will be used to measure the protein production of the component alpha chains of collagen IV and the isoforms of VEGF in response to high glucose and TGF-beta treatment. Whether high glucose and TGF-beta have synergistic effects will also be investigated. The cellular mechanisms underlying the high glucose-induced expression of the TGF-beta signaling receptor (preliminary data), which may explain the hypothesized high glucose/ TGF- beta synergy, will be elucidated by the nuclear runoff technique, analysis of promoter activity by luciferase reporter assay, and electrophoretic mobility shift assay. Lastly, specific inhibitors of TGF-beta will be used to determine whether some of the high glucose effects may be mediated by the TGF-beta system in the podocyte.